Tumor vaccine containing polysaccharide adjuvant and application thereof

Abstract

The invention relates to the field of biological medicine, and discloses a tumor vaccine containing a polysaccharide adjuvant and application of the tumor vaccine, and the tumor vaccine is prepared from, by mass, 100 parts of WT1-CM-beta G NPs, 10 parts of astragalus polysaccharide, 5 parts of saponin and 1 part of TLR agonist. The WT1-CM-beta G NPs is prepared from the following components in parts by mass: 10 parts of a WT1 polypeptide combination, 50 parts of carboxymethylated beta-glucan nano CM-beta G NPs, 10 parts of EDC (Ethylene Dichloride) and 6 parts of NHS (N-Hydroxysuccinimide). The WT1 polypeptide is covalently linked to the carboxymethylated beta-glucan nanoparticles through chemical coupling, then the WT1 polypeptide and the carboxymethylated beta-glucan nanoparticles cooperate with the adjuvant, the WT1 polypeptide and the carboxymethylated beta-glucan nanoparticles have a synergistic effect on improving the capability of stimulating DC maturation (highly expressing CD80 / CD86 / MHC-II) and improving the secretion of key Th1 type cell factors IL-12p70, can improve the anti-tumor effect, and have wide application in treating WT1 positive tumors.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, in particular to a tumor vaccine containing polysaccharide adjuvant and application thereof. BACKGROUND

[0002] Tumor therapeutic vaccine aims to activate the patient's own immune system to recognize and attack tumor cells. Its core components include tumor antigens and immune adjuvants. In recent years, natural polysaccharides have become a hot spot in the research of new adjuvants due to their unique immune regulation characteristics and good biological safety. However, in the current research and application, polysaccharide adjuvants are mostly used in the form of simple physical mixing of natural polysaccharides (such as lentinan and astragalus polysaccharide injection) with antigens. This form has obvious defects: antigens and adjuvants may not be distributed synchronously in vivo, which cannot ensure the uptake by the same antigen-presenting cells (APCs), and seriously limits the starting efficiency of immune response. Soluble polysaccharides and antigens are easily cleared by the body, lack of targeting, resulting in low lymph node accumulation, which requires large doses of frequent injection, not only increasing the cost but also possibly causing systemic inflammation risk. The mechanism research of many polysaccharide adjuvants still stays in the observation of overall phenomena (such as enhancing lymphocyte proliferation), lacking of in-depth understanding and engineering utilization of how to precisely regulate dendritic cell (DC) maturation, antigen cross-presentation and other key links. For tumor-associated antigens (TAA) such as WT1, the challenge is to break immune tolerance. The existing adjuvant system is often insufficient to efficiently achieve this, resulting in weak vaccine immunogenicity and limited clinical efficacy.

[0003] The market needs an "integrated intelligent immune activation platform" that integrates targeted delivery, synergistic activation and process optimization, and the WT1-CM-βG NPs of the present application solve such problems. SUMMARY

[0004] To solve the problems of the prior art, the present application aims to provide a tumor vaccine containing polysaccharide adjuvant and application thereof, which can realize targeted delivery, synergistic activation and process optimization.

[0005] In order to achieve the above-mentioned target, the technical scheme adopted by the present application is as follows: A tumor vaccine containing polysaccharide adjuvant, comprising, by mass fraction: 100 parts of WT1-CM-βG NPs, 10 parts of astragalus polysaccharide, 5 parts of quillaja saponin, and 1 part of TLR agonist; the WT1-CM-βG NPs comprise, by mass fraction: 10 parts of WT1 polypeptide combination, 50 parts of carboxymethylated β-glucan nanoparticles CM-βG NPs, 10 parts of EDC, and 6 parts of NHS.

[0006] The tumor vaccine containing polysaccharide adjuvant as described above, the WT1 polypeptide combination comprises, in terms of mass fraction: 2.5 parts of peptide segment 1, 2.5 parts of peptide segment 2, and 5.0 parts of peptide segment 3. The peptide segment 1 is: RMFPNAPYL SEQ ID No: 01. The peptide segment 2 is: CMTWNQMNL SEQ ID No: 02. The peptide segment 3 is: PGCNKRYFKLSHLQMHSRK SEQ ID No: 03.

[0007] The tumor vaccine containing polysaccharide adjuvant as described above, the carboxymethylated β-glucan nano CM-βG NPs comprise, in terms of mass fraction: 10 parts of yeast β-glucan CM-βG and 12 parts of sodium chloroacetate.

[0008] The tumor vaccine containing polysaccharide adjuvant as described above, the preparation method of the carboxymethylated β-glucan nano CM-βG NPs is as follows: 10 parts of yeast β-glucan is placed in a container, isopropyl alcohol is added as a dispersion medium, and is stirred and suspended; an alkali solution is added dropwise for alkalization, 12 parts of sodium chloroacetate is dissolved in ultrapure water, and is added dropwise to the reaction system; after warming and reaction, purification is performed to obtain yeast β-glucan CM-βG; and then an ultrasonic cell disrupter and a high-pressure homogenizer are used for treatment, and filtration is performed to obtain CM-βG nanoparticles CM-βG NPs.

[0009] The tumor vaccine containing polysaccharide adjuvant as described above, the preparation method of the WT1-CM-βG NPs is as follows: 50 parts of prepared CM-βG NPs is added with 10 parts of EDC and 6 parts of NHS to activate the carboxyl groups on the surface of the CM-βG NPs; then the WT1 polypeptide combination is added, the pH is adjusted to 6.5-7.5, and purification is performed after reaction to obtain the WT1-CM-βG NPs.

[0010] The tumor vaccine containing polysaccharide adjuvant as described above, the TLR agonist comprises one or a combination of several of imidazoquinoline (IMDQ), CpG oligodeoxynucleotides (CpG-ODN), Poly(I:C) (polyinosinic acid), LPS (lipopolysaccharide), or MPL (monophosphoryl lipid A).

[0011] The tumor vaccine containing polysaccharide adjuvant as described above, the TLR agonist is: Poly(I:C) (polyinosinic acid).

[0012] The tumor vaccine containing polysaccharide adjuvant as described above, the particle size of the WT1-CM-βG NPs is 100-200 nm.

[0013] The tumor vaccine containing the polysaccharide adjuvant according to the preceding, the particle size is 150 ± 20 nm, and the PDI is less than 0.2.

[0014] The tumor vaccine containing the polysaccharide adjuvant according to the preceding is applied to a carrier of a WT1-positive tumor therapeutic vaccine, a personalized neoantigen vaccine, a drug combined with a PD-1 / PD-L1 antibody, or an immune monitoring and diagnosis.

[0015] The present application has the advantages that: The present application covalently links the WT1 polypeptide to the carboxymethylated beta-glucan nanoparticle by chemical coupling, fundamentally solving the defects of physical mixing; this design ensures that the antigen and the adjuvant are phagocytosed by the APC as a whole, realizes precise 'co-delivery', greatly improves the efficiency of antigen processing and presentation, and is the key to inducing a strong T cell response.

[0016] The present application enhances lymph node targeting and accumulation: the polysaccharide is made into a nanoparticle of about 150 nm, which perfectly utilizes the physiological characteristics of the body's lymphatic system; particles of this size can be efficiently drained from the injection site to the lymph nodes and captured by a large number of DCs therein, thereby significantly reducing the dosage and reducing systemic exposure side effects.

[0017] The carboxymethylated beta-glucan nanoparticle and the quillaja saponin have a synergistic effect in improving the ability to stimulate DC maturation (high expression of CD80 / CD86 / MHC-II) and in improving the secretion of the key Th1-type cytokine IL-12p70, so that the formulation of the present application can improve the anti-tumor effect.

[0018] Carboxymethylation modification not only introduces carboxyl groups for chemical coupling, but also significantly improves the water solubility and stability of the beta-glucan preparation, making it more consistent with the basic requirements of industrialized drugs for raw materials.

[0019] Glossary: EDC (Ethyldimethylaminopropyl Carbodiimide), also known as 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, is an organic compound with the chemical formula C8H 17 N 3。

[0020] NHS (N-Hydroxy succinimide) N-hydroxy succinimide, white to white-like crystal.

[0021] Poly I:C (Polyinosinic-polycytidylic acid) is an artificial synthetic double-stranded ribonucleic acid, which is an interferon inducer and has antiviral and immunomodulatory functions. DETAILED DESCRIPTION

[0022] The application will be described in detail below with reference to specific embodiments.

[0023] Example 1: Formulation: 100 parts of WT1-CM-βG NPs, 10 parts of Astragalus polysaccharide, 5 parts of quillaja saponins, 1 part of TLR agonist; WT1-CM-βG NPs include, by mass fraction: 10 parts of WT1 polypeptide combination, 50 parts of carboxymethylated β-glucan nano CM-βG NPs, 10 parts of EDC, 6 parts of NHS. Carboxymethylated β-glucan nano CM-βG NPs include, by mass fraction: 10 parts of yeast β-glucan CM-βG, 12 parts of sodium chloroacetate.

[0024] The sample is prepared according to the following preparation method: Step one: carboxymethylation of yeast β-glucan; Principle: βG-OH + NaOH + ClCH2COONa → βG-O-CH2COONa + NaCl + H2O; Reaction system; Weigh 1.0 g of yeast β-glucan (YBG) into a 100 mL round-bottom flask; Add 20 mL of isopropyl alcohol as a dispersion medium and stir to suspend; Add 5 mL of 8 M NaOH solution dropwise, and alkalize at 30°C for 1 hour; Weigh 1.2 g of sodium chloroacetate (molar ratio: YBG repeat unit: ClCH2COONa ≈ 1:1.2), dissolve in 5 mL of ultrapure water, and slowly add to the reaction system; Increase the reaction temperature to 60°C and continue to react for 4 hours; Purification and post-treatment; After the reaction is completed, neutralize with 0.5 M HCl to pH ~ 7.0; Dialyze thoroughly with ultrapure water MWCO 3.5 kDa for 72 hours to remove residual salts, isopropyl alcohol and by-products; The product after dialysis is freeze-dried to obtain white flocculent solid CM-βG, which is stored at -20°C.

[0025] Step two: preparation of CM-βG nanoparticles; Prepare the solution: take 100 mg of CM-βG prepared in step one, dissolve in 20 mL of ultrapure water, and prepare a 5 mg / mL solution. Lightly heat or ultrasonic to aid dissolution; Pre-treatment: first use an ultrasonic cell disruptor (power 300W, on for 5s, off for 5s, total time 5min) for preliminary dispersion; High pressure homogenization: The solution was transferred to a high pressure homogenizer and homogenized 10 times at 15,000 psi under ice bath condition; Filtration: The homogenized suspension was filtered with 0.45 μm filter membrane to remove the possible small amount of large particles; Characterization: A small amount of sample was taken immediately and measured for hydrodynamic diameter and polydispersity index (PDI) by DLS. Target: particle size 150 ± 20 nm, PDI < 0.2.

[0026] Step three: Covalent coupling of WT1 polypeptides; Principle: EDC / NHS catalyzes the formation of amide bond between carboxyl and amino group; CM-βG-COOH + Pep-NH2 → CM-βG-CONH-Pep; Activation of carboxyl group: Take 10 mL of CM-βG NPs solution (5 mg / mL, total 50 mg CM-βG) prepared in step two; Add 10 mg EDC and 6 mg NHS (molar ratio EDC:NHS:COOH ≈ 2:1:1); Gently stir the reaction at room temperature for 15 minutes to activate the carboxyl group on the surface of CM-βG NPs.

[0027] Coupling of polypeptides: Weigh three kinds of WT1 polypeptides (peptide segment Pep1: 2.5 mg, peptide segment Pep2: 2.5 mg, peptide segment Pep3: 5.0 mg) and dissolve in 1 mL PBS (pH 7.4). (Molar excess of about 5-10 times); Peptide segment 1: RMFPNAPYL SEQ ID No: 01; Peptide segment 2: CMTWNQMNL SEQ ID No: 02; Peptide segment 3: PGCNKRYFKLSHLQMHSRK SEQ ID No: 03; Add the polypeptide solution drop by drop to the activated CM-βG NPs suspension; Carefully adjust the pH of the reaction system to 6.5-7.5 with 0.1 M NaOH; Gently react in the dark at room temperature for 4 hours; Purification; Transfer the reaction solution to a dialysis bag (MWCO 50 kDa) and dialyze against PBS (pH 7.4) for 24 hours, changing the solution every 4 hours to completely remove unreacted EDC / NHS and free polypeptides. The purified product is WT1-CM-βG NPs. Store at 4°C for short-term or freeze-dry for long-term storage.

[0028] Step four: Formulation of final vaccine preparation Formulation of adjuvant solution: 100 mg WT1-CM-βG NPs, 10 mg Astragalus polysaccharide, 5 mg Quillaja saponin QS-21, 1 mg Poly(I:C) were dissolved in 1 mL sterile PBS. Gently vortexed to mix, no need to filter. Final formulation was stored at 4°C away from light.

[0029] Step five, verification: Verification of biological activity with in vitro DC activation assay: co-incubation with mouse bone marrow derived DCs for 24 h, flow cytometry for CD80 / CD86 / MHC-II expression, ELISA for IL-12p70 secretion.

[0030] Comparative Example 1 Formulation: 100 parts WT1-CM-βG NPs, 5 parts Quillaja saponin, 1 part TLR agonist; WT1-CM-βG NPs included by mass fraction: 10 parts WT1 polypeptide combination, 50 parts carboxymethylated β-glucan nano CM-βG NPs, 10 parts EDC, 6 parts NHS. Carboxymethylated β-glucan nano CM-βG NPs included by mass fraction: 10 parts yeast β-glucan CM-βG, 12 parts sodium chloroacetate. Comparative Example 1 lacks Astragalus polysaccharide.

[0031] Comparative Example 2 Formulation: 100 parts WT1-CM-βG NPs, 10 parts Astragalus polysaccharide, 1 part TLR agonist; WT1-CM-βG NPs included by mass fraction: 10 parts WT1 polypeptide combination, 50 parts carboxymethylated β-glucan nano CM-βG NPs, 10 parts EDC, 6 parts NHS. Carboxymethylated β-glucan nano CM-βG NPs included by mass fraction: 10 parts yeast β-glucan CM-βG, 12 parts sodium chloroacetate. Comparative Example 1 lacks Quillaja saponin.

[0032] Comparative Example 3 Formulation: 100 parts WT1 polypeptide combination, 10 parts Astragalus polysaccharide, 1 part TLR agonist. Comparative Example 1 lacks carboxymethylated nanoparticles of β-glucan, lacks steps one to three, directly mixes WT1 polypeptide combination with adjuvants.

[0033] Comparative Example 4 Formulation: 100 parts of WT1-CM-βG NPs, 5 parts of quillaja saponin, 1 part of TLR agonist; WT1-CM-βG NPs according to mass fraction includes: 10 parts of WT1 polypeptide combination, 50 parts of yeast β-glucan CM-βG, 10 parts of EDC, 6 parts of NHS. Compared with Example 1, the β-glucan nanometer lacks carboxymethylation, and also lacks astragalus polysaccharide.

[0034] Comparative Example 5: Formulation: 100 parts of WT1-CM-βG NPs, 10 parts of astragalus polysaccharide, 1 part of TLR agonist; WT1-CM-βG NPs according to mass fraction includes: 10 parts of WT1 polypeptide combination, 50 parts of β-glucan nanometer βG NPs, 10 parts of EDC, 6 parts of NHS. Compared with Example 1, the β-glucan nanometer lacks carboxymethylation, and also lacks quillaja saponin.

[0035] Comparative Example 6: Formulation: 100 parts of WT1-CM-βG NPs, 10 parts of astragalus polysaccharide, 5 parts of quillaja saponin, 1 part of TLR agonist; WT1-CM-βG NPs according to mass fraction includes: 10 parts of WT1 polypeptide combination, 50 parts of β-glucan nanometer βG NPs, 10 parts of EDC, 6 parts of NHS. Compared with Example 1, the β-glucan nanometer lacks carboxymethylation.

[0036] Effect verification experiment: Experiment: WT1-CM-βG NPs nanometer vaccine in vitro dendritic cell (DC) maturation and activation performance evaluation.

[0037] I. Experimental materials and reagents Cell source: femur and tibia of 6-8 week old female C57BL / 6 mice; cytokines: recombinant mouse GM-CSF (rmGM-CSF), recombinant mouse IL-4 (rmIL-4).

[0038] Experimental grouping: Blank control group: complete RPMI-1640 culture medium; Positive control group: 1 μg / mL lipopolysaccharide (LPS, a classical DC strong activator); Experimental group 1: Example 1 formulation (containing APS, QS-21, TLR agonist, nanometer particles); Experimental group 2: Comparative Example 1 formulation (lacks astragalus polysaccharide); Experimental group 3: Comparative Example 2 formulation (lacks quillaja saponin); Experimental group 4: Comparative Example 3 formulation (physical mixing group); Experimental group 5: Comparative example 4 formula (β-glucan nanometer is used to lack carboxymethylation, and astragalus polysaccharide is also lacked); Experimental group 6: Comparative example 5 formula (β-glucan nanometer is used to lack carboxymethylation, and quillaja saponin is also lacked); Experimental group 7: Comparative example 6 formula (β-glucan nanometer is used to lack carboxymethylation); Adjuvant control group: blank CM-βG NPs (not coupled with polypeptide, containing TLR agonist); Flow cytometry antibodies: anti-mouse CD11c-APC, CD80-FITC, CD86-PE, MHC-II-PerCP-Cy5.5; ELISA kit: mouse IL-12p70 ELISA detection kit.

[0039] Instrument equipment: CO2 cell incubator, biological safety cabinet, flow cytometer, enzyme label instrument, centrifuge.

[0040] II. Experimental process Mouse bone marrow-derived DC (BMDC) was induced and cultured for 6 days. The femur and tibia of mice were taken aseptically, and the bone marrow cavity was washed with culture medium to obtain bone marrow cell suspension. Red blood cells were removed with red blood cell lysis solution, and the cells were resuspended with PBS. 2×10 6 cells / well were inoculated in a 6-well plate, and complete culture medium containing 20 ng / mL rmGM-CSF and 10 ng / mL rmIL-4 was used. Incubate in a 37°C, 5% CO2 incubator, half-volume medium replacement every two days, and supplement fresh culture medium containing cytokines.

[0041] Grouping and stimulation on the sixth day: immature BMDCs were collected, counted and resuspended. 1×10 6 cells / well were inoculated in a 24-well plate. According to the above grouping, the corresponding stimulants were added (all experimental groups were added according to the final concentration of WT1 polypeptide 5 μg / mL to ensure that the stimulation intensity was comparable). The cells were incubated in the incubator for 24 hours.

[0042] Day 7 sample collection and detection: Flow cytometry detection of surface molecules: collect cells in each well, wash twice with pre-cooled PBS. Add Fc blocking agent (anti-CD16 / 32 antibody) and incubate for 15 minutes to block non-specific binding. Add CD11c, CD80, CD86, MHC-II fluorescent antibody mixture according to the proportion in the instructions, avoid light for 30 minutes. Wash twice with PBS, resuspend in flow sample buffer. Detect on the machine, first circle the CD11c positive DC cell group, then analyze the average fluorescence intensity (MFI) and positive cell percentage of CD80, CD86, MHC-II in the group. The data results are shown in Table 1.

[0043] ELISA detection of cytokine secretion: At the same time of collecting cells, collect the cell culture supernatant in each well and store at -80°C. According to the operation instructions of the mouse IL-12p70 ELISA kit, detect the concentration (pg / mL) of IL-12p70 in each supernatant sample.

[0044] Four, experimental results and analysis 1. Flow cytometry results: Table 1

[0045] From the above experiments, it can be seen that the formula of the present application has the same effect as the strong activator LPS for CD80, CD86, and MHC-II in the group of cells, and even has a significantly better effect on MHC-II cells. From the comparison of the experimental results of Comparative Example 2 and Comparative Example 5, Comparative Example 6, it can be seen that the lack of QS-21 alone, and the use of unmethylated β-glucan nano, will make the DC cell activation effect not so high, but when both are lacking, the DC cell activation effect is significantly reduced. From the comparison of the experimental results of Comparative Example 1 and Comparative Example 4, Comparative Example 6, it can be seen that when both APS and methylated β-glucan nano are lacking, the DC cell activation effect is not significantly reduced, and is basically the same as the effect of lacking any one of them. Therefore, it can be seen that QS-21 and carboxymethylated β-glucan nano in the formula of the present application have a synergistic effect in improving the activation effect of DC cells.

[0046] The ELISA detection of cytokine secretion experimental results are shown in Table 2: Table 2

[0047] From the above experiments, the IL-12p70 secretion amount of Example 1 is as high as 1585.7 pg / mL, higher than the LPS positive control, and significantly higher than any other experimental group. From the comparison of the experimental results of Comparative Example 2 and Comparative Example 5, Comparative Example 6, it can be seen that the absence of QS-21 alone, and the use of unmethylated β-glucan nanoparticles, will make the DC cell activation effect not so high, but when both are absent, the DC cell activation effect is significantly reduced. From the comparison of the experimental results of Comparative Example 1 and Comparative Example 4, Comparative Example 6, it can be seen that when APS and methylated β-glucan nanoparticles are absent at the same time, the DC cell activation effect is not significantly reduced, and is basically the same as the effect of the absence of any one of them. It can be seen that the carboxymethylated nanoparticles of β-glucan and saponin (QS-21) have a synergistic effect in driving DC to secrete, and can maximize the driving of DC to secrete key Th1 polarized cytokines.

[0048] The above shows and describes the basic principles, main features and advantages of the present application. Those skilled in the art should understand that the above examples do not limit the present application in any form, and any technical solutions obtained by equivalent substitution or equivalent transformation fall within the protection scope of the present application.

Claims

1. A tumor vaccine containing a polysaccharide adjuvant, characterized in that, According to the mass fraction, it further includes: 100 parts of WT1-CM-βGNPs, 5 parts of quillaja saponins, and 1 part of TLR agonist; the WT1-CM-βGNPs according to the mass fraction include: 10 parts of WT1 polypeptide combination, 50 parts of carboxymethylated β-glucan nano CM-βG NPs, 10 parts of EDC, and 6 parts of NHS; the WT1 polypeptide combination according to the mass fraction includes: 2.5 parts of peptide segment 1, 2.5 parts of peptide segment 2, and 5.0 parts of peptide segment 3; the peptide segment 1 is: RMFPNAPYL SEQ ID No: 01; the peptide segment 2 is: CMTWNQMNL SEQ ID No: 02; and the peptide segment 3 is: PGCNKRYFKLSHLQMHSRK SEQ ID No:

03.

2. The tumor vaccine containing polysaccharide adjuvant according to claim 1, characterized by, According to the mass fraction, it further includes: 10 parts of astragalus polysaccharide.

3. A tumor vaccine containing a polysaccharide adjuvant according to claim 1 or 2, characterized in that, The carboxymethylated β-glucan nano CM-βG NPs according to the mass fraction include: 10 parts of yeast β-glucan CM-βG and 12 parts of sodium chloroacetate.

4. The tumor vaccine containing polysaccharide adjuvant according to claim 1 or 2, characterized in that, The preparation method of the carboxymethylated β-glucan nano CM-βG NPs is as follows: 10 parts of yeast β-glucan is placed in a container, isopropanol is added as a dispersion medium, and is stirred and suspended; an alkali solution is added dropwise for alkalization, 12 parts of sodium chloroacetate is dissolved in ultrapure water, and is added dropwise to the reaction system; after warming and reaction, purification is performed to obtain yeast β-glucan CM-βG; and after treatment with an ultrasonic cell disrupter and a high-pressure homogenizer, filtration is performed to obtain CM-βG nanoparticles CM-βG NPs.

5. The tumor vaccine containing polysaccharide adjuvant according to claim 4, characterized by, The preparation method of the WT1-CM-βGNPs is as follows: 50 parts of prepared CM-βG NPs is added with 10 parts of EDC and 6 parts of NHS to activate the carboxyl groups on the surface of the CM-βG NPs; WT1 polypeptide combination is further added, the pH is adjusted to 6.5-7.5, and after reaction, purification is performed to obtain WT1-CM-βGNPs.

6. The tumor vaccine containing polysaccharide adjuvant according to claim 1, characterized by, The TLR agonist includes one or a combination of several of imidazoquinoline (IMDQ), CpG Oligodeoxynucleotides (CpG-ODN), Poly(I:C) (polyinosinic acid), LPS (lipopolysaccharide), or MPL (monophosphoryl lipid A).

7. A tumor vaccine containing a polysaccharide adjuvant according to claim 6, characterized in that, The TLR agonist is: Poly(I:C) (polyinosinic acid).

8. The tumor vaccine containing polysaccharide adjuvant according to claim 1, characterized by, The particle size of the WT1-CM-βGNPs is 100-200 nm.

9. The tumor vaccine containing polysaccharide adjuvant according to claim 8, characterized by, The particle size is 150 ± 20 nm, and the PDI is less than 0.

2.

10. Use of a tumor vaccine containing a polysaccharide adjuvant according to claim 1, characterized in that, The carrier is used for a therapeutic vaccine for treating WT1-positive tumors, a personalized neoantigen vaccine, a drug for combination with a PD-1 / PD-L1 antibody, or for immune monitoring and diagnosis.

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